Flow cells and methods of filling and using same

ABSTRACT

Various flowcell configurations and systems are provided as are methods of making and using same. The flowcells, systems, and methods of use can be useful in carrying out sequencing reactions and next generation sequencing methods.

RELATED APPLICATIONS

This application claims priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61/238,633, filed on Aug. 31, 2009, entitled “Enhanced Systems and Methods For Sequence Detection,” U.S. Provisional Patent Application Ser. No. 61/238,667, filed on Aug. 31, 2009, entitled “Enhanced Flowcell and Reagent Delivery For Sequence Detection,” U.S. Provisional Patent Application Ser. No. 61/307,623, filed on Feb. 24, 2010, entitled “Methods of Bead Manipulation and Forming Bead Arrays,” U.S. Provisional Patent Application Ser. No. 61/307,492, filed on Feb. 24, 2010, entitled “Flowcells and Methods of Filling and Using Same,” U.S. Provisional Patent Application Ser. No. 61/307,641, filed on Feb. 24, 2010, entitled “Flowcells and Methods of Filling and Using Same,” and U.S. Provisional Patent Application Ser. No. 61/307,486, filed on Feb. 24, 2010, entitled “Flowcell, Flowcell Delivery System, Reagent Delivery System, and Method For Sequence Detection,” the entirety of each of these applications being incorporated herein by reference thereto.

FIELD

The present disclosure is directed towards molecular sequencing, in particular towards low-volume flowcell design and reagent delivery optimization.

BACKGROUND

There is a desire to make fluid handling systems more efficient in the use of reagents. There is also a desire to decrease volume requirements in fluid processing flowcells. To accomplish these objectives, requirements for fluid dispensing systems become more demanding. Some systems provide poor precision in low volume regimes, large dead volumes, and are of large overall size and plumbing lengths. With reduction in dimensions, however, it would become difficult to robustly and uniformly fill a flowcell without causing voids and bubbles. Furthermore, as surface area of the flowcell deposition surface increases, yield goes down and the cost of making the deposition substrate increases.

Lamination methods to make a flowcell using pressure sensitive adhesive (PSA) can be used at room-temperature, but factors, such as non-uniform PSA thicknesses and rough edges cut by die-cutting, may affect washing performance

SUMMARY

Various embodiment of a flowcell device are provided herein. For example, in one embodiment, the device can include a substrate with one or more flowcells formed in the substrate and each flowcell comprising a detection window. Additionally, the device can include a plurality of reagent supply reservoirs formed in the substrate with a plurality of fluid delivery pathways each in fluid communication with at least one of (a) the one or more flowcells, and (b) at least one of the plurality of reagent supply reservoirs, wherein each fluid delivery pathway is configured to be controlled by electro-wetting circuitry. The device can also include an electrical connector configured to provide electrical connections from a control unit to the electro-wetting circuitry. The substrate can include a plurality of layers. Further, the detection window can be transparent and include one or more of the layers.

Various embodiments of an analysis system are also provided herein. For example, the system can include an embodiment of the flowcell device and a control unit, wherein the control unit comprises an electrical connector configured to interface with the electrical connector of the flowcell device. The control unit can further include a detector configured to detect a reaction in each of the one or more flowcells when the electrical connector of the flowcell device is interfaced with the electrical connector of the control unit.

In another embodiment, a flowcell system is provided which includes a flowcell formed in a substrate, a first port in fluid communication with the flowcell, and a second port in fluid communication with the flowcell. Additionally, the system can include a reagent supply block comprising a plurality of reagent containers and a plurality of reagents disposed respectively in the plurality of reagent containers, and an injector system comprising one or more injectors disposed in an x-y-z movable stage with each injector comprising an injector tip. The system further includes a control system configured to move the injector system to the reagent supply block such that each injector tip can withdraw a reagent from the reagent supply block, and configured to move the injector system to the flowcell such that each injector tip can inject the reagent withdrawn from the reagent supply block through at least one of the first port and the second port and into the flowcell.

The one or more injectors can include a single injector or a plurality of injectors. The system can further include flexible tubing in fluid communication with the first port and the flowcell. In one embodiment, the first port includes a port formed in the substrate. The first port can include a zero volume injector port. Additionally, each of the first port and the second port can be in valved fluid communication with (i) a source of vacuum, (ii) a source of air or argon gas, (iii) a source of wash solution, and (iv) a waste reservoir. In one embodiment, each of the first port and the second port is in fluid communication with a rotary valve that in turn is in fluid communication with each of (i) the source of vacuum, (ii) the source of air or argon gas, (iii) the source of wash solution, and (iv) the waste reservoir.

Various embodiments of a method of making a flowcell are also provided herein. For example, in one embodiment, the method includes forming channel-defining features on a surface of a substrate, and adhesively bonding a cover layer to the channel-defining features to form a flowcell comprising at least one channel, wherein the adhesively bonding comprises curing a uv-curable adhesive. In one embodiment, the uv-curable adhesive can include a mercapto-ester adhesive. The channel-defining features can be formed from, for example, a uv-curable adhesive, and the channel-defining features can include a mercapto-ester adhesive.

In one embodiment, the adhesively bonding step includes spin-coating a uv-curable adhesive layer onto the cover layer and then contacting the uv-curable adhesive layer with the channel-defining features. Additionally, in one embodiment, the adhesively bonding step can include positioning the cover layer adjacent the channel-defining features to form a capillary gap there between, filling the capillary gap with a uv-curable adhesive, by capillary action, and curing the uv-curable adhesive. In one embodiment, the forming channel-defining features on a surface of a substrate step includes applying a layer of uv-curable adhesive to the surface of the substrate, masking portions of the layer of uv-curable adhesive layer, curing portions of the uv-curable adhesive layer that are not masked, by exposure to uv-radiation, and cleaning off portions of the uv-curable adhesive layer that were not masked and that were not cured, leaving channel-defining features formed on the surface of the substrate.

Various embodiments of a method of making a flowcell are provided herein. In one embodiment, the method includes laminating a die-cut or laser-cut adhesive film onto a surface of a substrate to form channel-defining features on the surface of the substrate, and laminating a cover layer to the channel-defining features to form a flowcell comprising at least one channel In one embodiment, the die-cut or laser-cut adhesive film comprises a die-cut pressure sensitive adhesive layer. In one embodiment, the die-cut or laser-cut adhesive film includes a laser-cut polyimide film. Additionally, in one embodiment, the cover layer can include a cyclo-olefin polymer material.

In one embodiment, the method can further include forming one or more fluidic inlets to the at least one channel, wherein the one or more fluidic inlets are formed through the substrate. The method can further include disposing reagents in the flowcell and contacting the cover layer with a heating surface of a thermal cycler. In one embodiment, the method can further include disposing reagents in the flowcell and contacting the substrate with a heating surface of a thermal cycler.

Various embodiments of a method filling a flowcell are also provided herein. In one embodiment, the method includes introducing a reagent into a first end of a flowcell, using a pressurized pump, the flowcell comprising a channel defined at least in part by a glass slide and a thermal black. While introducing the reagent, the method can also include drawing a vacuum on an opposite end of the flowcell to pneumatically facilitate filling the flowcell with the reagent.

In one embodiment, the flowcell can include a tapered configuration having a narrower end at the first end and a wider end at the opposite end. The flowcell can include a support between the thermal block and the glass slide, the support being configured to maintain a desired spacing between the thermal block and the glass slide.

Various embodiment of a flowcell are also provided. In one embodiment, the flowcell includes a first substrate having a first surface, the first substrate comprising a thermal block, an alternating electrode band layer having a surface facing a surface of the thermal block, and a polymeric coating on a surface of the alternating band layer opposite the surface facing the thermal block, wherein the polymeric coating defines the first surface. The flowcell can also include a second substrate comprising a second surface facing the first surface, the second substrate comprising a glass slide having a reaction surface comprising a plurality of reaction sites thereon, the reaction surface defining the second surface, and a flow channel defined between the first surface and the second surface. In one embodiment, the reaction surface comprises a plurality of beads fixed to a surface of the glass slide. In one embodiment, the flowcell further comprises a printed circuit board between the thermal block and the alternating electrode band layer.

Various embodiments of a flowcell system are also provided herein. For example, in one embodiment the system includes a flowcell, a flowcell supply pathway in fluid communication with the flowcell, and a flowcell supply pathway degassing system configured to degas gas from the flowcell supply pathway before the gas reaches the flowcell.

In one embodiment, the system can further include a detector positioned along the flowcell supply pathway and configured to detect gas in the flowcell supply pathway. The system can further include an aspirator tip in fluid communication with the flowcell supply pathway and configured to aspirate gas in the flowcell supply pathway before the gas reaches the flowcell. In one embodiment, the flowcell system can further include a porous hydrophobic material defining at least a portion of the flowcell supply pathway and configured to degas gas in the flowcell supply pathway by gas venting, before the gas reaches the flowcell. In one embodiment, the flowcell system can further include an air bubble injector in fluid communication with the flowcell supply pathway and configured to inject air bubbles as spacers between reagent volumes flowing through the flowcell supply pathway.

Various embodiments of a method of mixing different reagents together in a flowcell supply pathway are also provided herein. In one embodiment, the method includes injecting an air bubble into a flowcell supply pathway of an embodiment of a flowcell system disclosed herein. The method further includes injecting different reagents into the flowcell supply pathway, behind the air bubble, then injecting another air bubble into the flowcell supply pathway, behind the different injected reagents, and moving the air bubbles and the different injected reagents through the flowcell supply pathway to cause mixing of the different injected reagents in the flowcell supply pathway.

Various embodiments of a flowcell system are also provided herein. In one such embodiment, the system includes a flowcell, a flowcell supply pathway in fluid communication with the flowcell, and a mixing chamber disposed along the flowcell supply pathway and in fluid communication with a plurality of different reagent supplies. The system can also include a flowcell syringe pump configured to move fluid from the mixing chamber into and through the flowcell.

In one embodiment, the flowcell system can further include an injection manifold assembly comprising the plurality of different reagent supplies, and a feed line in fluid communication with the mixing chamber, wherein each of the different reagent supplies of the plurality of different reagent supplies is in valved communication with the feed line. In one embodiment, the system, can also include a multiplex manifold assembly comprising the plurality of different reagent supplies, a feed line in fluid communication with the mixing chamber, and a pressure source, wherein each of the different reagent supplies of the plurality of different reagent supplies is in valved communication with the feed line, and the pressure source is configured to force the different reagent supplies of the plurality of different reagent supplies into the feed line.

In one embodiment, a flowcell system is provided which includes a flowcell, a flowcell supply pathway in fluid communication with the flowcell, and a reagent supply disposed along the flowcell supply pathway and in fluid communication with the flowcell supply pathway. The system can also include a system fluid supply disposed along the flowcell supply pathway and in fluid communication with the flowcell supply pathway, and a regulated air pressure source configured to pump reagent from the reagent supply into and through the flowcell supply pathway and configured to pump system fluid from the system fluid supply into and through the flowcell supply pathway.

In one embodiment, the system can include a syringe pump configured to pull system fluid from the system fluid supply and move the system fluid into the flowcell supply pathway. The system can also include a second reagent supply disposed along the flowcell supply pathway and in fluid communication with the flowcell supply pathway, a first syringe pump configured to pull system fluid from the system fluid supply and move reagent from the reagent supply into the flowcell supply pathway, and a second syringe pump, different than the first, configured to pull system fluid from the system fluid supply and move second reagent from the second reagent supply into the flowcell supply pathway.

These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a side view of a detection system that can be used to detect signals generated in a flowcell, according to various embodiments of the present teachings;

FIG. 2 is a substrate with electrodes configuration for an electro-wetting fluid manipulation system according to various embodiments of the present teachings, wherein electrodes define a deposition region and a number of buses to transport reagents of interest from reservoirs to specific deposition regions;

FIG. 3 is a top view of an integrated card device according to various embodiments of the present teachings, wherein reagent reservoirs, fluid delivery and mixing paths, and flowcells are all integrated into the device;

FIG. 4 is an exploded view of a system according to various embodiments of the present teachings, showing a sample slide, a plurality of reagents in a chiller block, and a multi-injector apparatus;

FIG. 5 is an exploded view of a system according to various embodiments of the present teachings, showing a sample slide, a plurality of reagents in a chiller block, and a zero-volume multi-injector apparatus;

FIG. 6 is an exploded view of a system according to various embodiments of the present teachings, showing a sample slide, a plurality of reagents in a chiller block, and a single injector apparatus;

FIG. 7 is an exploded view of a system according to yet other various embodiments of the present teachings, showing a sample slide, a plurality of reagents in a chiller block, and a single injector apparatus;

FIG. 8 is a diagram of a system according to various embodiments of the present teachings, showing a sample slide and a single injector fluid manipulation system comprising rotator valves;

FIG. 9 is a side view of a channel layer for a microchannel device according to yet other various embodiments of the present teachings;

FIG. 10 is a flow diagram of two steps of a manufacturing method that uses the channel layer of FIG. 9 and a spin-coated adhesive cover layer, wherein the diagram shows side views of a cover layer, an adhesive-coated cover layer, and an assembled microchannel device according to various embodiments of the present teachings;

FIG. 11 is a flow diagram of two steps of a manufacturing method that uses the channel layer of FIG. 9 and a cover layer adhered thereto by an adhesive applied by a capillary application method, wherein the diagram shows side views of a cover layer, an adhesive-application step, and an assembled microchannel device according to various embodiments of the present teachings;

FIG. 12 is a flow diagram of three steps of a manufacturing method to make a channel layer according to various embodiments of the present teachings, wherein an adhesive is applied by spin-coating and uv light and a mask are used to form a channel layer for a microchannel device;

FIG. 13 is a flow diagram of a step of spin-coating a glass slide with a uv-curable adhesive to form a cover layer according to various embodiments of the present teachings;

FIG. 14 is a side view of an assembled microchannel device comprising the channel layer of FIG. 12 and the cover layer of FIG. 13, according to various embodiments of the present teachings;

FIG. 15 is a flow diagram of two steps of a lamination manufacturing method useful to make an assembled microchannel device according to various embodiments of the present teachings;

FIG. 16 is a side view of a laminating step of a manufacturing method to form an assembled microchannel device according to various embodiments of the present teachings;

FIG. 17 is a side view of a fluidics interface device comprising a slide and a zirconium coating, according to various embodiments of the present teachings;

FIG. 18 is a side view of the fluidics interface device of FIG. 17, showing the device positioned with respect to a heating apparatus and an optics system, according to various embodiments of the present teachings;

FIG. 19 is a side view of the fluidics interface device of FIG. 17, showing the device positioned with respect to a heating apparatus and an optics system, according to other embodiments of the present teachings;

FIG. 20 is a side view of a flowcell comprising a tapered assembly configured to be filled using a vacuum assist system, according to various embodiments of the present teachings;

FIG. 21 is a side view of a flowcell comprising a slide support and configured to be filled using a vacuum assist system, according to various embodiments of the present teachings;

FIG. 22 is a side view of a flowcell assembly configured to be pressure filled using a fluidic pump, according to various embodiments of the present teachings;

FIG. 23 is a side view of a flowcell assembly configured to be filled using electro-wetting, according to various embodiments of the present teachings;

FIG. 24 is a side view of a degassing system for degassing a reagent pathway for a flowcell, according to various embodiments of the present teachings;

FIG. 25 is a schematic diagram of a degassing system comprising a pathway along a porous member, to a flowcell, according to various embodiments of the present teachings;

FIG. 26 is a side view of an air bubble aspirator system for aspirating an air bubble from a reagent pathway for a flowcell, according to various embodiments of the present teachings;

FIG. 27 is a schematic diagram of an air bubble aspirating system comprising an air bubble detector and aspirator along a reagent pathway to a flowcell, according to various embodiments of the present teachings;

FIG. 28 is a schematic diagram of a reagent injection pumping system for pumping reagents to a flowcell, according to various embodiments of the present teachings;

FIG. 29 is a schematic diagram of a multiplexed reagent injection pumping system for pumping reagents to a flowcell, according to various embodiments of the present teachings;

FIG. 30 is a schematic diagram of a pressure-driven air bubble injection system to promote flow mixing in a flowcell, according to various embodiments of the present teachings;

FIG. 31 is a schematic diagram of a reagent injection pumping system for pumping reagents to a flowcell, according to various embodiments of the present teachings;

FIG. 32 is a schematic diagram of a reagent injection pumping system for pumping reagents to a flowcell, according to various embodiments of the present teachings.

DETAILED DESCRIPTION

According to various embodiments of the present teachings, a fluid handling system is provided that comprises a multi-axis pipetting robot, a syringe pump, a distribution valve, tubing, a Peltier-based reagent chiller, and a Peltier flowcell heater. The components can be off-the-shelf and fluidic processing protocols using the system can be flexible for a variety of reagents, can be flexible with respect to changing assays, and can provide robust mixing and a relatively low cost for hardware. In some embodiments, many existing components of the Life Technologies SOLiD platform (Life Technologies, Carlsbad, Calif.) can be used. In some embodiments, a flowcell is provided for the Life Technologies SOLiD platform. In one embodiment, an embodiment of a flowcell as described in Applicant's co-pending U.S. Patent Application Ser. No. ______, filed on Aug. 31, 2010, entitled “Low-Volume Sequencing System and Method of Use,” the entirety of which is incorporated herein by reference thereto.

According to various embodiments, the flowcell can comprise a system wherein molecules of interest are attached to a transparent substrate and are observed with an objective. The surface that has the attached molecules can at least partially define an enclosed volume into which various reagents can be introduced to make contact with the molecules of interest. FIG. 1 is a side view of a detection system that can be used to detect signals generated in the flowcell.

In FIG. 1, a flowcell 40 is shown which comprises a fluidic chamber 42 in which reagents can be reacted with molecules of interest attached to the inner surface 44 of a transparent bead substrate 46, for example, a glass slide. In the embodiment shown, the molecules of interest can be colonized on beads 48 that have been deposited on and fixed to inner surface 44. A gasket seal 50 can be provided to seal fluidic chamber 42. A thermal block 52 can be provided, for example, as shown, to partially define fluidic chamber 42.

Flowcell 40 can be mounted vertically as shown, or horizontally, or in a diagonal orientation. A pressure driven flow system can be used to fill the flowcell 40, and gravity can be used to assist in filling and draining the pressure driven flow system. The depth of the flowcell can be less than about 300 times the depths of the viewed features and 10 or less multiples of the diffusion layers. Dimensions can be minimized to minimize the volume of reagents that are used. In some embodiments, the depth of the flowcell is reduced relative to the depth of existing SOLiD platform flowcells. The flowcells and flowcell systems according to the present teachings overcome challenges that would otherwise arise with a reduced flowcell depth. In some embodiments, signals from multiple fields of view are detected one field at a time. The signals can be those emitted for a volume of one of a multiple fields of view.

According to various embodiments, a massively high throughput system is provided that comprises a large surface area. In some embodiments, a system is provided that scans an entire field after each stepwise addition of nucleic acid bases in a sequencing reaction. The scanning can occur in a fixed location and a synchronous addition of bases can be implemented.

In some embodiments, a system is provided to control the hydrophobic and hydrophilic nature of surfaces of the flowcell by means of an electro-wetting system and process. As exemplified in FIG. 2, a substrate 54 comprising a multiple-electrode configuration. An electro-wetting fluid manipulation system according to various embodiments of the present teachings is provided, wherein electrodes define a plurality of deposition regions 56 and a number of buses 58 to transport reagents of interest from reservoirs to specific deposition regions 56. Depending on electrode density, smaller portions of deposition regions 56 can be divided to form sub-portions 60 of a size and shape desired.

According to various embodiments, a hybrid system is provided wherein a mixture of small electrodes and electrode pads of larger surface areas can be combined to create preconfigured regions. The large electrodes can be used to define deposition regions, and the small electrodes can each be used as a bus to transport reagents of interest from a reservoir to a specific deposition region.

In some embodiments, a method is provided that uses electro-wetting properties and an arrangement as exemplified in FIG. 2 to uniformly fill and evacuate a wide channel having a shallow depth. The flowcell electro-wetting system can be provided that utilizes capacitive charging forces to uniformly fill and evacuate imaging areas with controlled precision.

In some embodiments, a system is provided that comprises one electrode and a ground point or plane. Charging can be used to change the nature of wetting of a flowcell for facilitating filling. In a one-dimensional system, alternating electrode bands can be provided to enable charging of flowcell reagents for controlled filling and evacuation. In a two-dimensional embodiment, even greater flexibility in terms of flowcell filling can be provided. Specific flowcell regions can be targeted and customized deposition geometries can be enabled. The protocols can be optimized to have imaging, for example, scanning, synchronized with targeted flowcell filling. Such a desired configuration can enable synchronous scanning of bases in a single field of view in embodiments where the system divides detection of the flowcell into multiple fields of view. Reagent delivery can be targeted to a single field of view at a time. Each field of view can be fluidically isolated for reagent delivery. Chip surfaces can be decreased in size for better yield, within practical limits. By utilizing a system of targeted reagent delivery system and method as described herein, high throughput levels can be achieved.

In some embodiments, methods to improve reaction and exchange efficiency in a flowcell are provided. Electrodynamic movement of fluid can be used to improve the efficiency. During a period of contact between a reagent and molecules of interest within the flowcell, for example, during incubation or a nucleic acid incorporation event, mixing can be facilitated by alternating electrode electro-wetting charges to create movement of the reagents and thus improve reactivity by promoting greater uniform exposure of the molecules of interest to the reagents. Exposure to the residual reagents left behind after evacuation can be minimized as well. In some embodiments, this reduces the amount of reagents and time needed for subsequent wash steps.

According to various embodiments, an integrated on-chip system is provided that comprises an electro-wetting configuration of electrodes and deposition regions. The electro-wetting system can be scaled to incorporate all the fluid handling subsystems at a low cost. An exemplary embodiment is shown in FIG. 3, which is a top view of an integrated chip 64. The chip configuration shown illustrates exemplary components of a chip comprising reagent reservoirs in the form of wells 66, fluid delivery paths 68, fluid mixing areas 69, and flowcells 70 all integrated into a card-shaped substrate 72. Chip 64 can comprise a cover layer 74, for example, a transparent film, on top of substrate 72. Chip 64 can be configured to be plugged into an electrical control and detection system (not shown). This configuration can enable a zero dead volume system that can manipulate nanoliter-sized volumes. Chip 64 can be loaded with stored bulk reagents that are deposited into wells 66, and the reagents can then be distributed, as needed, in any desired quantity. A simple electrical edge connector 76 can be used for the electrical interfaces with the electrical control and detection system. Registration points, holes, or other features such as a registration corner 78 can be provided to optically couple the flowcells to the detector, camera, and/or scanner.

In some embodiments, the integrated chip and system can enable very low overall reagent consumption and can enable precise sub-microliter dispensing of expensive sequencing probe reagents. The system and method provide efficient exchange rates and reactivity of molecules. The overall foot print can be compact and small. Very little, if any, tubing is required and the chip can be easy to manufacture. The system and method provide small dead volumes and little wasted reagents, and enable high throughput for synchronous single molecule sequencing platforms. Also, the system and method enable a reuse of small sample volumes in the microliter size range. The concepts can be applied to the Life Technologies (Carlsbad, Calif.) SOLiD platform and fluidic handling subsystems including next generation SOLiD and single molecule sequencing systems.

According to various embodiments, yet another flowcell system configuration is provided. The flowcell system exhibits improved fluid handling relative to existing flowcell systems, such that the flowcell can be better washed, for example, between reagent delivery steps. The system provides a minimum volume pathway of reagents from a sample tray, for example, from an autosampler tray, to a flowcell slide, and thus reduces reagent usage and washing effort. In some embodiments, reagents can efficiently be added from an autosampler tray to the slide and can be efficiently returned back to the autosampler tray.

In some embodiments, a current autosampler for a Life Technologies (Carlsbad, Calif.) SOLiD platform is modified to attach a single injector or multi-injector to the xyz arm. Reagents can be aspirated from the autosampler tray vial using an existing syringe pump. In some embodiments, the sample can be either aspirated into the syringe barrel or into a sample loop having a hold volume large enough to retain the desired reagent volume. The device can be used to replace the current plumbing system in the SOLiD platform. The injector configuration can eliminate any need for reagent to traverse from a reagent vial, through an autosampler needle, through a syringe valve block, and through a long length of tubing on its way to the slide. Retrieval of the reagent from slide back to the autosampler tray can be provided by reversing the addition process.

The shorter pathway can be much less complicated to clean between reagent delivery steps, relative to longer existing pathways. Also, the injector/sample loop plumbing can be cleaned using wash solutions pumped from a large wash solution reservoir and into a liquid waste station. An exemplary configuration is shown schematically in FIG. 8, described in more detail below, which is a diagram of a system according to various embodiments of the present teachings, showing a sample slide and a single injector fluid manipulation system comprising rotator valves.

In some embodiments, the injectors can be configured to deliver wash solutions via a large reservoir to wash the slide. The injectors can be pressurized or pump-assisted. The single injector or multi-injector can deliver air and/or argon, for example, to assist in removing liquids using a vacuum source. Also, the slide can be washed without the injector using a source of wash solution connected to the slide. While washing the slide is described herein, it is to be understood that it is the flow slide defined by the slide, which is cleaned and loaded with reagents.

The injector or injectors can be mated to and/or otherwise align with ports of the slide so that delivery of reagents can be made from the top or the bottom of the slide. The configuration can enable a reduction in bubbles formed during filling the slide and can improve washing of the slide.

FIG. 4 is an exploded view of a system 80 according to various embodiments of the present teachings. System 80 comprises a flowcell slide apparatus 82, a plurality of reagents in a multi-compartment chiller block 84, and a multi-injector apparatus 86. Flowcell slide apparatus 82 comprises plumbing described below and a slide 83. Slide 83 defines a flowcell having an inlet 88 and an outlet 90. Flexible tubes 92 and 94 can be provided in fluid communication with inlet 88 and outlet 90, respectively. Flexible tubes 92 and 94 can enable sample to move as the tubes and flowcell are filled. Injector ports 96 and 98 and respective vales 100 and 102 are provided to respectively control filling and evacuating of the flowcell defined by slide 83. Injector ports 96 and 98 can comprise zero volume ports that are, for example, fixed and in close proximity to the multi-injector apparatus 86 so multi-injector apparatus 86 needs only to move a short distance to be aligned for injection. Inlet 88 and outlet 90 can be provided with valves as shown. Waste reservoirs 104 and 106 can be provided in fluid communication with injector ports 96 and 98 respectively.

As shown in FIG. 4, the plurality of reagents in the multi-compartment chiller block 84 can be segregated in a plurality of respective compartments 108. Multi-injector apparatus 86 can comprise a plurality of injector tips 110 that are configured to move reagents from chiller block 84 to ports 96 and 98 of slide apparatus 82. Injector tips 100 can be mounted in an xyz stage 112. Injector tips 110 can be in fluid communications with a conduit 114 configured to interface with a syringe, a conduit 116 configured to interface with a wash solution reservoir, a conduit 118 configured to interface with a source of air and/or argon, and a conduit 120 configured to interface with a vacuum source.

FIG. 5 is an exploded view of a system 122 according to various embodiments of the present teachings, showing a zero-volume injector port slide apparatus 124, a plurality of reagents in a chiller block 84, and a multi-injector apparatus 86. Zero-volume injector port slide apparatus 124 comprises plumbing described below and a slide 83. Slide 83 defines a flowcell having an inlet 88 and an outlet 90. Injector ports 96 and 98 and respective vales 101 and 103 are provided to respectively control filling and evacuating of the flowcell defined by slide 83. Injector ports 96 and 98 can comprise zero volume ports that are, for example, fixed and in close proximity to the multi-injector apparatus 86 so multi-injector apparatus 86 needs only to move a short distance to be aligned for injection. Waste reservoirs 104 and 106 can be provided in fluid communication with injector ports 96 and 98 respectively.

As shown in FIG. 5, the plurality of reagents in the multi-compartment chiller block 84 can be segregated in a plurality of respective compartments 108. Multi-injector apparatus 86 can comprise a plurality of injector tips 110 that are configured to move reagents from chiller block 84 to ports 96 and 98 of slide apparatus 124. Injector tips 100 can be mounted in an xyz stage 112. Injector tips 110 can be in fluid communications with a conduit 114 configured to interface with a syringe, a conduit 116 configured to interface with a wash solution reservoir, a conduit 118 configured to interface with a source of air and/or argon, and a conduit 120 configured to interface with a vacuum source.

FIG. 6 is an exploded view of a system 126 according to various embodiments of the present teachings. System 126 comprises an xyz movable flowcell slide apparatus 128, a plurality of reagents in a multi-compartment chiller block 84, and a single injector apparatus 130. Flowcell slide apparatus 128 comprises plumbing described below and a slide 83. Slide 83 defines a flowcell having an inlet 88 and an outlet 90. Flexible tubes 92 and 94 can be provided in fluid communication with inlet 88 and outlet 90, respectively. Flexible tubes 92 and 94 can enable sample to move as the tubes and flowcell are filled. Injector ports 96 and 98 and respective vales 100 and 102 are provided to respectively control filling and evacuating of the flowcell defined by slide 83. Injector ports 96 and 98 can comprise zero volume ports that are, for example, fixed and in close proximity to the single injector apparatus 130 so single injector apparatus 130 needs only to move a short distance to be aligned for injection. Waste reservoirs 104 and 106 can be provided in fluid communication with injector ports 96 and 98 respectively.

As shown in FIG. 6, the plurality of reagents in the multi-compartment chiller block 84 can be segregated in a plurality of respective compartments 108. Single injector apparatus 130 can comprise a first injector tip 132 that is configured to move reagents from chiller block 84 to ports 96 and 98 of slide apparatus 128. First injector tip 132 and second injector tip 134 can be mounted in an xyz stage 136 and can be in fluid communications with a first conduit 138 and a second conduit 140, respectively. First conduit 138 can be in fluid communication with a sample loop and a syringe. Second conduit 140 can be configured, for example, with a rotary valve, to be in selective fluid communication with a wash solution reservoir 142, a source of air and/or argon 144, a vacuum source 146, and a waste reservoir 148. Valves can provided between conduit 140 and each of the sources with which it is in fluid communication. Injector tips 132 and 134 can be at different heights. Injector tips 132 and 134 can share the same stage.

FIG. 7 is an exploded view of a system 150 according to yet other various embodiments of the present teachings, showing a sample slide apparatus 152, a plurality of reagents in a chiller block 86, and a single injector apparatus 154. Sample slide apparatus 152 comprises plumbing described below and a slide 83. Slide 83 defines a flowcell having an inlet 88 and an outlet 90. Conduits 156 and 158 can be provided in fluid communication with inlet 88 and outlet 90, respectively. Conduits 156 and 158 can enable sample to move as the flowcell is filled. Injector ports 96 and 98 and respective vales 100 and 102 are provided to respectively control filling and evacuating of the flowcell defined by slide 83. Injector ports 96 and 98 can comprise zero volume ports that are, for example, fixed and in close proximity to the multi-injector apparatus 86 so single injector apparatus 154 and/or slide 83 need only to move a short distance to be aligned for injection. Waste reservoirs 104 and 106 can be provided in fluid communication with injector ports 96 and 98, respectively, for example, through respective rotary valves. One or more vacuum source 120 can be provided in fluid communication with injector ports 96 and 98, respectively, for example, through respective rotary valves. One or more air and/or argon sources can be provided in fluid communication with injector ports 96 and 98, respectively, for example, through respective rotary valves. One or more wash solution reservoirs can be provided in fluid communication with injector ports 96 and 98, respectively, for example, through respective rotary valves.

As shown in FIG. 7, the plurality of reagents in the multi-compartment chiller block 84 can be segregated in a plurality of respective compartments 108. Single injector apparatus 154 can comprise an injector tip 132 that is configured to move reagents from chiller block 84 to ports 96 and 98 of sample slide apparatus 152. Injector tip 132 can be mounted in an xyz stage 136 and can be in fluid communications with a conduit 138. Conduit 138 can be in fluid communication with a sample loop and a syringe.

FIG. 8 which shows an exploded schematic view of a system according to various embodiments of the present teachings, showing a sample slide 160 in a system comprising many of the features described above with reference to FIGS. 4-7. An autosampler 161 is also shown along with a sample tray 169 from which sample can be withdrawn and injected into sample slide 160. Autosampler 161 can comprise a single injector needle injector 165 operated by a syringe 163 and a rotary valve 167. FIG. 8 depicts a rotary valve 162 in fluid communication with inlet 88 and many other sources as described herein. FIG. 8 also depicts a rotary valve 164 in fluid communication with outlet 90 and many other sources as described herein. Rotary valves 162, 164, and 167 can each independently be in fluid communications with a conduit configured to interface with a syringe 163, a conduit configured to interface with a wash solution reservoir 116, a conduit configured to interface with a source of air and/or argon 118, and a conduit configured to interface with a vacuum source 120. Rotary valves 162, 164, and 167 can each independently be in fluid communication with a conduit configured to interface with an auxiliary source 170, for example, a source of buffer, other reagent, pressure differential, or the like. Rotary valves 162, 164, and 167 can each independently be in fluid communication with a conduit configured to interface with a waste conduit or reservoir 104, 106, or 172, respectively.

The systems shown in FIGS. 4-8 minimize contamination and loss of reagents caused by dead volume. The systems also enable an efficient washing of a flowcell and minimize reagent volume usage by using short pathways and by returning uncontaminated reagent for reuse.

According to various embodiments of the present teachings, a fabrication method is provided to make a flowcell. The configuration minimizes cost per run for sequencing reactions using the flowcell. The configuration also reduces run time of a sequencing reaction using the flowcell, for example, when used in conjunction with the SOLiD platform available from Life Technologies of Carlsbad, Calif.

According to various embodiments, a system comprising a microfluidics channel is provided that can manipulate a very small volume of liquid, for example, a volume of 25 uL or less, for example, of about 10 uL or less. The system provides fast delivery of reagents and sample to a flowcell and efficiently positions reagents in the flowcell. In some embodiments, a uv-curable adhesive is used in the construction of a flowcell according to the present teachings. In an exemplary embodiment, NORLAND adhesive, a uv-curable adhesive from Norland Products, is used in manufacturing a multi-layered flowcell according to the present teachings. The refractive index of the NORLAND adhesive can approximate that of silica glass, and it has very low auto-fluorescence. Exemplary adhesives are NORLAND's optical adhesives, NOA 60 (a mercapto-ester adhesive), NOA 61 (a mercapto-ester adhesive), NOA 63 (a mercapto-ester adhesive), NOA 65 (a mercapto-ester adhesive), and NOA 68 (a mercapto-ester and tetrahydrofurfuryl methacrylate adhesive). UV-curable glues with these properties can be used in various embodiments. After fully curing, NORLAND adhesive exhibits no detrimental effect on enzymes or oligonucleotides used, for example, in a PCR or other DNA sequencing reaction. The adhesive can be PCR-compatible. Various NORLAND adhesives generally used for glass bonding, plastics bonding, and/or glass-plastic bonding, can be used.

In an exemplary embodiment, as shown in FIG. 9, a channel layer 180 can be bonded to a cover layer by spin-coating of the adhesive, by capillary action placement of the adhesive, by another adhesive moving force, or by a combination thereof. In FIG. 9, it can be seen that channel layer 180 comprises a substrate 182 and channel defining structures, here in the form of walls 184. In some embodiments, NORLAND adhesive itself can be used to build up a structure that defines a feature of channel layer 180, a cover layer, an intermediate layer, or a combination thereof. The viscosity of the NORLAND is small enough to enable the adhesive to be moved by capillary action into a very small gap, for example, into a sub-micrometer (μm) gap. Channel layer 180 can be fabricated from glass and by a method comprising glass etching, channel build up by SU-8, a combination thereof, and the like. Channel layer 180 can be fabricated from plastic and by a method comprising machining, molding, a combination thereof, and the like.

FIG. 10 shows a method of spin-coating a uv-curable adhesive, for example, a NORLAND adhesive, into a layer 186 on a glass slide 188 to form a cover layer 190. Subsequently, cover layer 190 is bonded to channel layer 180 to form a multi-channel flowcell 192. Bonding can be by contacting channel layer 180 with cover layer 190 and curing adhesive layer 186 with ultraviolet (uv) light.

FIG. 11 shows a method of applying a uv-curable adhesive, for example, a NORLAND adhesive, as a droplet 187. The viscosity and properties of the adhesive enable it to be moved by capillary action into a sub-micrometer gap formed between channel layer 180 and a glass slide 188 that have been brought together. For this purpose, an exemplary NORLAND adhesive that can be used is NOA 89 (a low viscosity, mercapto-ester and 1,6 hexanediol diacrylate adhesive). The capillary action causes the adhesive to move in the direction shown by the arrowhead between glass slide 188 and channel layer 180. The adhesive can form a layer that can be cured to bond glass slide 188 to channel layer 180 to form a multi-channel flowcell 194.

Bonding a channel layer to a cover layer can be achieved by any suitable method, but a spin coating method is depicted in FIG. 10 and a capillary action spreading method is depicted in FIG. 11. As shown in FIGS. 12-14, the spinning speed for the adhesive coating can be used to determine the height of the channel, for example, to define a microfluidic channel.

FIGS. 12-14 show a method of manufacturing a multi-channel flowcell 200 shown in FIG. 14. Flowcell 200 comprises a channel layer 202 made the method depicted in FIG. 12, and a cover layer 204 made by the method depicted in FIG. 13. As shown in FIG. 12, an ordered array, for example, available as a plastic or cyclic olefin, or a glass slide 206 comprising a metal-oxide layer, is spin-coated with a uv-curable adhesive to form an adhesive layer 208 on glass slide 206. A mask 210 is applied to adhesive layer 208 and the resulting structure 212 is exposed to uv-light 214. The uncured adhesive is then removed leaving channels defined by the cured adhesive.

As shown in FIG. 13, cover layer 204 is formed by spin-coating an adhesive layer 216 onto a glass slide 218. Cover layer 204 is then contacted with channel layer 202 and uv light 220 is used to cure adhesive layer 216.

Yet other embodiments of the present teachings are shown in FIGS. 15-19. A fabrication method to construct a flowcell, and a flowcell system, are provided. The flowcell reduces the cost per run for a sequencing reaction, reduces the run time for a sequencing reaction, and is cheaper, simpler, and more reliable than existing flowcells. In some embodiments, lamination of pressure sensitive adhesive (PSA) at room-temperature provides a simple and fast method of making a microfluidics channel structure, for example, a flowcell.

As shown in FIG. 15, an ordered array, for example, available as a plastic or cyclic olefin, or a glass slide 206 comprising a metal oxide coating, is laminated with an adhesive layer or film, such as a cut adhesive layer, for example, a die-cut or laser-cut adhesive layer 222. Die-cutting or laser-cutting can be used to define channel walls 224 in the resulting flowcell structure. Die-cut or laser-cut adhesive layer 222 can be very thin, for example, less than 100 μm in thickness. Exemplary materials for die-cut or laser-cut adhesive film 222 include, but are not limited to, pressure sensitive adhesive materials, polyimide materials, and the like. Adhesive film 22 can then be laminated with a cover layer 226 to form a flowcell 228. Cover layer 226 can comprise a polymeric material, or the like, for example, a cyclic olefin polymer material, or the like, and can be very thin, for example, less than 100 μm in thickness.

FIG. 16 shows a method of laminating cover layer 226 to a channel layer 230 by using the nip 232 between two rotating nip rollers 234 and 236.

A fluidics interface can be provided for a flowcell such as flowcell 228 shown in FIG. 15. As mentioned, the thickness of a PSA layer can be used to determine the height of a microfluidics channel through flowcell 228, which can be, for example, less than 100 μm. FIG. 17 shows that fluidics inlets and outlets to the microfluidic channels of a flowcell can be formed by making through-holes 238 in glass slide 206 or substrate layer, of flowcell 228 shown in FIG. 15, to form an assembly 240. In the case of plastics, through holes 238 can be drilled, molded, or otherwise machined, and in the case of a glass substrate ultrasonic drilling can be used to provide a very clean and smooth through-hole.

Fluidic inlet and/or outlet through holes 238 can be interfaced with an instrument, for example, with a reagent supply system, by o-ring, septum, an elastic material, a combination thereof, and the like. The fluidics interface can comprise a gasket layer with thru-holes formed therein. The gasket can be clamped against the flowcell, the instrument, or both. In some embodiments, the gasket fluidics interface is encapsulated in a fluidics interface assembly and fluidics interface assembly can comprise a gasket layer on one side against the slide or substrate, and a fluidics interface with the robot tip at the other side.

FIGS. 18 and 19 depict how the laminated package can be interfaced with thermal and optical systems in the instrument, and show the pros and cons of each orientation. In the configuration shown in FIG. 18, an optical pathway is directed toward the brighter side of beads disposed in the flowcell. In FIG. 18, the flatness of cover film 226 does not affect the flatness of image taken of the flowcell. In the embodiment of FIG. 19, the relatively thin layer of cover film 226, compared to the thickness of substrate 206, provides better thermal conduction compared to the embodiment of FIG. 18.

In yet other embodiments of the present teachings, efficient use is made of reagents to minimize amounts needed. The fluid handling system manipulates many reagents and takes into consideration accuracy, precision, residual carryover, contamination, cost, speed, head pressure, storage conditions and temperature, dispensing resolution, mixing capability, chemistry protocols, and uniform filling of the flowcells without voids. Exemplary configurations and approaches for implementation of a high performance fluid handling system that takes these factors into consideration are shown in connection with FIGS. 20-32.

FIGS. 20-22 show embodiments of the present teachings that enable effective flowcell filling. In some embodiments, a pneumatic system is used with or without a pumping system. FIG. 20 shows a flowcell system 250 comprising a tapered design and that uses vacuum assist to load reagents. Flowcell system 250 comprises a flowcell 252 defined between a glass slide 254 and a thermal block 256. Dispensed reagent 258 is pulled into flowcell 252 through a syringe pump fill port 260 at the bottom of flowcell 252, and vacuum 262 applied to the top of flowcell 252 facilitates liquid loading of flowcell 252.

FIG. 21 shows a flowcell system 251 comprising a configuration that uses vacuum assist to load reagents. Flowcell system 251 comprises a flowcell 253 defined between a glass slide 254 and a thermal block 256. Dispensed reagent 258 is pulled into flowcell 253 through a syringe pump fill port 260 at the bottom of flowcell 253, and vacuum 262 applied to the top of flowcell 253 facilitates liquid loading of flowcell 253. A support 264 is provided in a central portion of flowcell 253 to maintain the shape of flowcell 253 during vacuum loading of sample and reagents. Support 264 can be configured to maintain a separation distance between glass slide 254 and thermal block 256.

FIG. 22 shows a flowcell system 266 comprising a flexible slide 268 and a valve or port configuration that enables pressure to be built up inside a flowcell 270 defined between flexible slide 268 and a thermal block 256. A fluidic pump filling system 272 can be provided, for example, having an opening to flowcell 270 at a bottom or bottom portion of flowcell 270. Another opening 274 can be provided, for example, at a top or top portion of flowcell 270, for example, for introducing sample or reagent and/or for plugging flowcell 270 so that pressure can be built up inside by fluidic pump filling system 272. Relaxation of flexible slide 268 after pressurization can be used as a motive force enabling movement of reagents into and through flowcell 270.

In some embodiments, a system 280 and method of filling a flowcell can involve an electro-wetting configuration as shown in FIGS. 23A-23C. System 280 offers fast fluid handling performance and enables a flow through system. System 280 comprises a glass slide 282 having a plurality of reaction sites and/or beads 284 deposited thereon and defining a surface of a flowcell 286. Flowcell 286 is also defined by a polytetrafluoroethylene coating 288 on an electrode layer 290 comprising a plurality of electrode bands 292 (FIG. 23C). FIG. 23C is an enlarged view of portion 2300 shown in FIG. 23B. Electrode bands 292 can be deposited on a PC board 292 that is in contact with a thermal block 294. With the flow through system, disposed reagents 296 can follow each other through flowcell 286 with an air gap between each different reagent, eliminating the need to evacuate and/or drain flowcell 286 before each subsequent reagent loading step. The new reagents replace the previous reagents with very little or no carryover. The method reduces the number of wash steps and makes efficient use of reagents.

According to various embodiments, a fluid handling system is provided that efficiently uses reagents by enabling a relatively fast diffusion of reagents wherein the diffusion layer thickness is smaller compared to the height of a flowcell. The present teachings maximize the efficient incorporation of reagents. Alternating electrode bands 292 of electrode layer 290 enable electro-wetting allows and a recirculation loop that assists in diffusion of reagent molecules to analytes of interest at the reaction sites and/or beads. Electrode bands or other electro-wetting configurations of electrodes can be used to enable an effective means of agitation of reagents within the flowcell. In some embodiments, reagents are exposed to ambient conditions that allow for evaporation of reagents effectively increasing the concentration of the reagents being consumed.

According to various embodiments, challenges with current designs are overcome by providing an air gap between reagents sequentially loaded into a flowcell. In some embodiments, as shown in FIGS. 24 and 25, a degassing system 300 is provided that comprises an air bubble 302 or air gap that moves fluids through a flowcell entry pathway 304 formed, at least in part, by a manifold body 320. Manifold body 320 can comprise or be in contact with a porous hydrophobic membrane 322, for example, a gas venting membrane. Pressure driven flow 306 can be provided by a source to drive various reagents, for example, reagents 308, 310, and 312, through flowcell entry pathway 304 on their way to a flowcell 305. The degassing system enables the use of air bubbles and air spacers while also enabling a method to degas the air so that it does not reach the flowcell. FIG. 25 shows a degasser 322 comprising a long pathway along a porous hydrophobic membrane 322.

According to various embodiments, FIGS. 26 and 27 demonstrate a degassing system 330 that comprises an air bubble aspirator 332 configured to aspirate air bubbles between reagent aliquots sequentially loaded and flowed through a flowcell entry pathway 334 on their way to a flowcell 335. In some embodiments, degassing system 330 comprises an air bubble 302 or air gap that moves fluids through flowcell entry pathway 334 formed, at least in part, by a manifold body 336. Manifold body 336 can comprise or be in contact with air bubble aspirator 332. Pressure driven flow 306 can be provided by a source to drive various reagents, for example, reagents 308, 310, and 312, through flowcell entry pathway 334 on their way to flowcell 335. FIG. 27 shows air bubble aspirator 332 used in conjunction with, and timed with, a detector 338 along flowcell entry pathway 334. The aspirating system enables the use of air bubbles and air spacers along a flowcell entry pathway while also enabling a method to degas the air so that it is removed and does not reach the flowcell.

In some embodiments, the system can automatically remove air bubbles that may have inadvertently been introduced, in addition to removing intentionally introduced spacing air gaps. The system can also detect and remove air bubbles resulting from the breakup of intentionally injected air gaps. Gentle aspiration can be used to achieve a robust removal of air bubbles while minimizing reagent mixing in the flowcell.

The present teachings provide a system and method for sensing and monitoring reagent streams to and/or from a flowcell. The streams can be monitored for air bubbles and the system can be configured to aspirate air bubbles from the stream. The system and method can completely remove air bubbles while minimizing the amount of excess reagents aspirated and preventing reagent carryover.

Further embodiments of the present teachings include those depicted with reference to FIGS. 28-32. In some embodiments, reagent indexing and delivery is provided by a pipetting robot that is versatile, accurate, and efficient with small volume dispensing. In some embodiments, however, a system is configured for moving reagents with an automated injection system. The systems described with reference to FIGS. 28-32 incorporate aspects of an injection manifold system with the flexibility and accuracy of a syringe pump. The system can replace conventional pipetting and instead can use an injection valve manifold. In some embodiments, 3-port, 2-way valves are provided that are connected in series on a manifold that comprises a common port connecting the valves. The syringe pump allows for versatility in dispensing volumes. Two exemplary embodiments are shown in FIGS. 28 and 29.

FIG. 28 is a schematic diagram of a reagent injection pumping system for pumping reagents to a flowcell, according to various embodiments of the present teachings. FIG. 28 shows a system comprising reagent injection pumping with dedicated valves. As shown in FIG. 28, a system 350 is provided wherein an injection manifold assembly 352 allows for zero dead volume, thus preserving reagents, and a syringe pump 354 allows for precise metering of reagents. One valve 353 can be provided for each different reagent. In some embodiments, reagents that are to be mixed can be grouped into the same manifold such that carryover and cross contamination are of very little or no concern. In embodiments where carryover is of concern, a wash cycle can be added to clean the fluid lines. System 350 can comprise a mixing chamber 356, a flowcell 358, and a waste reservoir 360. In some embodiments, the pumping method is indirect, so the reagents do not actually traverse the pump and distribution valve and the system avoids problems of large dead volumes associated with the use of too many valves.

FIG. 29 is a schematic diagram of a multiplexed reagent injection pumping system for pumping reagents to a flowcell, according to various embodiments of the present teachings. FIG. 29 shows a system comprising reagent injection pumping and multiplexed valves. The embodiment enables a low cost approach to minimizing the number of valves by multiplexing reagents. FIG. 29 shows an exemplary schematic of a multiplexed valve array system 370 comprising an injection manifold assembly 372 that allows for zero dead volume, thus preserving reagents, and a syringe pump 374 allows for precise metering of reagents. Valves 376 can be provided, one for each reagent column and one for each reagent row. Reagents that are to be mixed can be grouped into the same manifold such that carryover and cross contamination are of very little or no concern. System 370 can comprise a mixing chamber 378, a flowcell 380, and a waste reservoir 382. In some embodiments, the pumping method is indirect, so the reagents do not actually traverse the pump and distribution valve and the system avoids problems of large dead volumes associated with the use of too many valves.

In some embodiments, a multiplexed system is provided wherein there is one valve for each row of reagents and reagent columns are addressed by another set of valves. For example, to address 25 reagents, 10 valves are needed (five for five rows and five for five columns) The column valves can be configured to enable pressurization or vacuum release, enabling a particular reagent to be pulled out preferentially to other reagents in the row.

In some embodiments, effective reagent mixing can be performed by adding all the reagents of interest into a mixing chamber. Then the contents of the chamber can be exposed to several cycles of aspiration and dispensing. Another method to mix reagents is to introduce reagents into a channel under laminar flow conditions trapped between air bubbles. As the slug moves along the channel under pressure driven flow, the contents are mixed due to the fluidic drag of the side walls. FIG. 30 illustrates the effect.

FIG. 30 is a schematic diagram of a pressure-driven air bubble injection system, and an enlarged close-up of a portion thereof, useful to promote flow mixing along a pathway to a flowcell, according to various embodiments of the present teachings. As shown in FIG. 30, a flowcell entry pathway 390 is in fluid communication with an air bubble injector 392 comprising a valve 394. Three reagent sources 396, 398, and 400 provide three different respective reagents to flowcell entry pathway 390. After an injection of a first air bubble 402 into entry pathway 390, all three reagents are then injected to entry pathway 390 followed by injection of a second air bubble 404. Movement of air bubbles 402 and 404, and the reagents between the air bubbles, through entry pathway 390, causes mixing currents 406 as demonstrated in the enlarged close-up portion of FIG. 30. Mixing currents 406 can promote mixing of the three reagents as they travel through entry pathway 390 on their way toward a flowcell (not shown).

Among the advantages achieved by a system as shown in FIG. 30, the components are simple, the system is compact and has a small overall footprint, there is an ability to address many reagents, there are small reagent delivery volumes that can be dispensed precisely, there is less tubing than existing fluid supply pathways, the system is easy to manufacture, there is less dead volume and wasted reagents compared to known systems, there are shorter fluidic pathways that require less washing than longer pathways, and there is an increase in intermolecular interaction efficiency. Other advantages include no crystallization of solids on sliding seals, less time to test effects of chemicals on pumps, less volume to switch fluids such as rinse solutions and reagents, and less cross-contamination of fluids. These teachings can be applied to platforms that use fluidic handling subsystems, for example, including the SOLiD platform from Life Technologies of Carlsbad, Calif.

In yet another embodiment of the present teachings, FIG. 31 shows a schematic diagram of a reagent injection pumping system for pumping reagents to a flowcell. A system 410 is shown wherein a regulated air pressure source 412 provides regulated air pressure to a deionized system fluid source 414 and to a chemical source 416 such as a source of a reagent. Flow restrictors 418 and 420 can be provided as well as injection valves 422 and 424, to control injection of air, reagents, and system fluid along a pathway 426 to a flowcell. A syringe pump 428 can also be used to control the flow of fluids in the correct direction and rate, through pathway 426.

FIG. 32 is a schematic diagram of a reagent injection pumping system for pumping reagents to a flowcell, according to yet other embodiments of the present teachings. A system 430 is shown wherein a regulated air pressure source 432 provides regulated air pressure to a deionized system fluid source 434 and to chemical sources 436, 438, 440, and 442, such as sources of different respective reagents. Flow restrictors can be provided as well as injection valves to control injection of air, reagents, and system fluid along pathways to a flowcell. A plurality of syringe pumps 448 can also be used to control the flow of fluids in the correct direction and rate, through the pathways. The system can comprise a source of elution buffer 450 driven by a syringe pump 458 to a dispensing pipette 460.

One skilled in the art will appreciate further features and advantages of the present disclosure based on the above-described embodiments. Accordingly, the disclosure is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety. 

1. A flowcell device comprising: a substrate; one or more flowcells formed in the substrate, each flowcell comprising a detection window; a plurality of reagent supply reservoirs formed in the substrate; a plurality of fluid delivery pathways each in fluid communication with at least one of (a) the one or more flowcells, and (b) at least one of the plurality of reagent supply reservoirs, wherein each fluid delivery pathway is configured to be controlled by electro-wetting circuitry; and an electrical connector configured to provide electrical connections from a control unit to the electro-wetting circuitry.
 2. The flowcell device of claim 1, wherein the substrate comprises a plurality of layers and the detection window is transparent and comprises one or more of the layers.
 3. A system comprising the flowcell device of claim 1 and a control unit, wherein the control unit comprises an electrical connector configured to interface with the electrical connector of the flowcell device.
 4. The system of claim 3, wherein the control unit further comprises a detector configured to detect a reaction in each of the one or more flowcells when the electrical connector of the flowcell device is interfaced with the electrical connector of the control unit.
 5. A flowcell system, comprising: a flowcell formed in a substrate; a first port in fluid communication with the flowcell; a second port in fluid communication with the flowcell; a reagent supply block comprising a plurality of reagent containers and a plurality of reagents disposed respectively in the plurality of reagent containers; an injector system comprising one or more injectors disposed in an x-y-z movable stage, each injector comprising an injector tip; and a control system configured to move the injector system to the reagent supply block such that each injector tip can withdraw a reagent from the reagent supply block, and configured to move the injector system to the flowcell such that each injector tip can inject the reagent withdrawn from the reagent supply block through at least one of the first port and the second port and into the flowcell.
 6. The flowcell system of claim 5, wherein the one or more injectors comprises a single injector.
 7. The flowcell system of claim 5, wherein the one or more injectors comprises a plurality of injectors.
 8. The flowcell system of claim 5, further comprising flexible tubing in fluid communication with the first port and the flowcell.
 9. The flowcell system of claim 5, wherein the first port comprises a port formed in the substrate.
 10. The flowcell system of claim 5, wherein the first port comprises a zero volume injector port.
 11. The flowcell system of claim 5, wherein each of the first port and the second port is in valved fluid communication with (i) a source of vacuum, (ii) a source of air or argon gas, (iii) a source of wash solution, and (iv) a waste reservoir.
 12. The flowcell system of claim 11, wherein each of the first port and the second port is in fluid communication with a rotary valve that in turn is in fluid communication with each of (i) the source of vacuum, (ii) the source of air or argon gas, (iii) the source of wash solution, and (iv) the waste reservoir.
 13. A method of making a flowcell, comprising: forming channel-defining features on a surface of a substrate; and adhesively bonding a cover layer to the channel-defining features to form a flowcell comprising at least one channel, wherein the adhesively bonding comprises curing a uv-curable adhesive.
 14. The method claim 13, wherein the uv-curable adhesive comprises a mercapto-ester adhesive.
 15. The method claim 13, wherein the channel-defining features are formed from a uv-curable adhesive.
 16. The method claim 15, wherein the channel-defining features comprise a mercapto-ester adhesive.
 17. The method claim 13, wherein the adhesively bonding comprises spin-coating a uv-curable adhesive layer onto the cover layer and then contacting the uv-curable adhesive layer with the channel-defining features.
 18. The method claim 13, wherein the adhesively bonding comprises: positioning the cover layer adjacent the channel-defining features to form a capillary gap there between; filling the capillary gap with a uv-curable adhesive, by capillary action; and curing the uv-curable adhesive.
 19. The method of claim 13, wherein the forming channel-defining features on a surface of a substrate comprises: applying a layer of uv-curable adhesive to the surface of the substrate, masking portions of the layer of uv-curable adhesive layer; curing portions of the uv-curable adhesive layer that are not masked, by exposure to uv-radiation; and cleaning off portions of the uv-curable adhesive layer that were not masked and that were not cured, leaving channel-defining features formed on the surface of the substrate. 